The need for a purified preparation of hemoglobin as a starting material for use as a resuscitation fluid was first recognized by Rabiner, Journal of Exp. Med., 126, 1127 (1967). He was able to remove a large amount of the stromal elements and demonstrate that most of the toxic properties of hemolyzed red cells were related to the membranes (stroma) and associated lipids. Subsequent to his demonstration that hemoglobin was compatible as an additive into the intravascular system, many workers modified his initial production scheme. Doczi, U.S. Pat. No. 3,991,181, discloses a method which makes extensive use of centrifugation to wash the red blood cells with various saline solutions prior to hemolysis in hypotonic phosphate buffers. He then removes the stromal contamination by ultrafiltration on an 0.22.mu. filter, dialyzes the filtrate to achieve crystalloid composition compatible with physiological systems, and filters the solution through a stack of filters 8.mu. to 0.22.mu. to remove protein aggregates and bacteria thus sterilizing the filtrate prior to use or storage. It is in fact pertinent to realize that the process simply has removed the membrane from the red cell and has kept the entire contents of the red cell including a 5% by weight non-heme protein.
Bonson et al., U.S. Pat. No. 4,001,401, described a method using washed rbc's, hypotonic lysis, centifugation and repeated centifugation after toluene extraction. They also use dialysis to gain crystalloid physiological compatibility. Again the final solution contains the entire content of the erythrocyte. DeVenuto et al. continued the prior art of Bonson et al. by adding the additional step of crystallization from concentrated Hb solution (14%) in 2.8 M potassium phosphate pH 6.8 buffer. This was the first method that attempted to remove non-heme protein from the stroma-free hemoglobin solution. Using many techniques it is easy to show that crystallization does not remove non-heme protein efficiently and repeat recrystallization does not increase the purity.
Another significant problem exists in the task of collecting outdated red blood cells from hospitals and blood banks throughout the United States and the world, for use in the production of a hemoglobin based blood substitute. If the outdated blood must be washed at the collection center and/or stored and refrigerated prior to shipping on a bimonthly basis, compliance for collection will be decreased. Alternately, if the outdated blood could be frozen and shipped when sufficient units were collected to warrant collection, the compliance would be greater. None of the methods mentioned above for isolating hemoglobin have been used to isolate hemoglobin from hemolyzed blood containing serum proteins and unwashed red blood cells.
Recently Carrell and Lehmann, J. Clin. Path., 34, 796 (1981) and Lehmann et al., Li Hemoglobin, 6(2), 183-186 (1982) reported the use of zinc ion to precipitate unstable and normal hemoglobins from dilute solutions between analytical methodologies. The precipitated hemoglobin was incubated at 37.degree. C. for 15 min., collected by centrifugatio and resolubilized by EDTA.
A need exists for a method to purify hemoglobin in large quantities, free of stroma, serum proteins and non-heme intraerythrocytic proteins. Minimal standards for such hemoglobin were set by a committee of U.S. Army researchers preparing stroma-free hemoglobin for modification and use as a blood substitute. Only hemoglobin made by the process of this invention meet these standards which follows:
(a) non-heme protein--&lt;0.1% PA1 (b) stromal elements--undetectable PA1 (c) lipids--&lt;0.001% PA1 (d) endotoxin--pass LAL PA1 (1) Purification of hemoglobin from the non-heme proteins within the erythrocyte PA1 (2) concentration of hemoglobin from dilute solutions PA1 (3) purification and concentration of hemoglobins from other sources such as bovine PA1 (4) purification and concentration of hemoglobins after modification PA1 (5) purification and concentration of hemoglobin from plasma proteins PA1 (6) recycling of hemoglobin through the procedure to obtain ultrapure material for clinical or analytical standards.
Thus hemoglobin can be prepared from either frozen whole blood or preferably washed cells hemolyzed in hypotonic buffer used to make the initial stroma-free Hb.